Daniel L. Dexter

Heterogeneity of cancer cells from a single human colon carcinoma

The human colon carcinoma cell line DLD-1, established from tumor tissue obtained from a 45 year old white man with an adenocarcinoma of the sigmoid colon, was studied from the perspective of tumor heterogeneity. The karyotype and morphology of cells from an early passage DLD-1 culture, as well as the histologic features of both the original tumor and neoplasms produced by inoculation of athymic nude mice with DLD-1 cells, indicated that both the DLD-1 cell line and the original tumor were heterogeneous. Two clones were isolated from the DLD-1 line; they differed in their morphology, karyotype, and cloning efficiency in soft agar. Furthermore, when cells from each clone were injected into athymic mice, histologically distinct tumors were produced. Various analyses showed that the two cloned lines were representative of the two subpopulations predominantly responsible for the heterogeneity of the original neoplasm. In vitro drug screening results demonstrated that the two cloned lines have differential sensitivities to chemotherapeutic agents. The parent DLD-1 human colon carcinoma cell line and its two cloned subpopulations provide material for the study of various aspects and implications of human cancer cell heterogeneity.

Clinical and pharmacological implications of cancer cell differentiation and heterogeneity

Abstract Various aspects and interrelationships of intra-neoplastic diversity have been investigated at two levels: (1) differences among multiple subpopulations of tumor cells at any point in time (heterogeneity) and (2) variations among these subpopulations as a result of their maturation with time (differentiation). The importance of tumor cell heterogeneity and differentiation in neoplasia has been studied using cancer cells obtained from murine mammary tumors and rhabdomyosarcoma, as well as from patients with carcinoma of the colon. Several cancer cell lines derived from these tumors have been cloned and characterized. Neoplastic cell differentiation has been induced using a polar solvent, N,N-dimethylformamide (DMF); the differentiation is evidenced by morphological maturation, conversion of tumor cell markers and cell culture characteristics to those consistent with a more benign phenotype, and loss of tumorigenicity. Striking morphological and biological heterogeneity has been observed in neoplastic subpopulations isolated from a single tumor, including marked variability in growth potential, surface antigens, and sensitivity to chemotherapeutic agents. The biological and pharmacological significance of these findings is discussed from the standpoint of their profound implications for clinical therapy.

Maturation‐induction of tumor cells using a human colon carcinoma model

The establishment of a maturation‐induction model using human colon cancer cells as targets is reported. Two colon carcinoma cell lines were established from human tumors; one line was heterogeneous and was cloned into two distinct subpopulations. Cells from these lines and clones and cells from an established human colon carcinoma cell line were treated in vitro with N, N‐dimethylformamide (DMF) and were compared to untreated cells according to two general sets of criteria. One set contains characteristics that define a cell as transformed (i.e., anchorage independence and tumorigenicity for nude mice), and the second set contains three antigenic marker systems that would provide evidence that maturation is occurring in treated human colon cancer cells. These colon tissue‐or tumor‐related markers include carcinoembryonic antigen (CEA), colonic mucoprotein antigen (CMA), and the human blood group determinants. DMF‐treated cells are less malignant than untreated cells; the treated cells show a marked reduction in tumorigenicity and in clonogenicity in soft agar. Each of the markers indicates that the treated cells are better differentiated than their untreated counterparts. For example, treated cells show increased expression of normal‐CMA and decreased expression of tumor‐CMA compared to untreated cells. Cells removed from DMF reverted to express the tumorigenicity, growth properties, and antigens characteristic of their untreated counterparts. Therefore, DMF reversibly induces in cultured colon cancer cells a less malignant phenotype with concomitant maturational effects. These results indicate that this model is appropriate for study of maturation‐induction in human colon tumor cells, and has potential application to other types of human carcinomas.

Isolation and characterization of an undifferentiated human colon carcinoma cell line (MIP-101).

An undifferentiated human colon carcinoma cell line was established from tumor tissue obtained from metastasis to the liver of colonic adenocarcinoma in a patient with fulminant Dukes D colorectal carcinoma. Histological analysis of the tumor biopsy from the liver confirmed the hospital pathology report of poorly differentiated colonic adenocarcinoma. Explants of this tumor tissue xenografted into a nude mouse were used to establish an epithelioid-like cell culture line, MIP-101. The cell line formed tumors in nude mice that histologically appeared undifferentiated and did not stain for carcinoembryonic antigen (CEA). No CEA was present either by radioimmunoassay (RIA) of the culture supernatant or by immunoperoxidase staining of the tumors or monolayers. MIP-101 appears to be one of the most undifferentiated human colon carcinoma cells lines available. It should prove useful in the search for markers of undifferentiated colonic cancer and in studies of colonic cancer differentiation.

Sodium butyrate-induced alteration of growth properties and glycogen levels in cultured human colon carcinoma cells

SummaryThe effect of sodium butyrate on three cultured human colon carcinoma cell lines was studied. Exposure to butyrate caused morphological changes and resulted in the alteration of several growth properties. Doubling times of treated cells were increased five-fold and saturation densities and cloning efficiencies were decreased. compared to untreated cells. Histochemical studies using the periodic acid-Schiff reaction in conjunction with diastase digestion showed that butyrate induced increased glycogen levels in all three cell lines. This increase was confirmed by biochemical techniques. These effects of butyrate were reversed when treated cells were subsequently grown in the absence of butyrate. These changes are consistent with findings from several laboratories that butyrate can induce, phenotypic changes in cultured tumour cells.

Chemotherapy of cell‐line‐derived human colon carcinomas in mice immunosuppressed with antithymocyte serum

An in vivo model is described for assessing the antitumor activity of chemotherapeutic agents. Tumors derived from human colon carcinoma cell lines injected into antithymocyte serum (ATS) immunosuppressed mice were used. In this system, both antitumor effects and host toxicity can be quantitated, permitting calculation of a Therapeutic Index. Compared with other xenograft models, the present system is simple, experiments are completed in less than 2 weeks, and the use of cultured cell lines allows in vitro studies to be performed. The in vitro sensitivities of one colon cell line to 22 chemotherapeutic agents and of four cell lines to three agents is reported. Four drugs used in treating colon cancer (Mitomycin C, 5‐FU, BCNU, and methyl‐CCNU) show antitumor activity in vivo in this system. Each has a low therapeutic index. Further work with this model is indicated, with the goal of finding new drugs with high Therapeutic Indices.

Ultrastructural evidence of dimethylformamide-induced differentiation of cultured human colon carcinoma cells increased expression of desmosomes

N,N‐dimethylformamide (DMF) induces differentiation of human colon carcinoma (DLD‐1) cells in culture and reduces their tumorigenicity in nude mice. The current investigation analyzed DLD‐1 (clone D) cells for ultrastructural evidence of differentiation. Examination of treated and untreated confluent monolayers by transmission electron microscopy revealed an occasional intracytoplasmic lumen indicative of adenocarcinoma. DMF‐treated cells showed no signs of a toxic reaction. Cytoplasmic organelles were essentially unchanged except for an increase in tonofilaments and associated desmosomes. The number of desmosomes per unit length of contiguous cell border increased almost sixfold in treated monolayers. No other type of cell junction was seen. The increased frequency of desmosomes in DMF‐treated cultures is significant because of the direct correlation known to exist between the number of desmosomes and degree of differentiation of some human carcinomas. Desmosomes serve as foci of cell adhesion and are reduced in number in some invasive tumors. Whether the supernumerary desmosomes in DMF‐treated cells contribute to the reduction in malignant behavior of these cells in vivo remains to be determined. Cancer 56: 1559‐1556, 1985.

Selective modification of the X ray survival response of two mouse mammary adenocarcinoma sublines by N,N-dimethylformamide

Abstract The modification of the X ray survival response of two mammary adenocarcinoma tumor cell sublines by the polar solvent N,N-dimethylformamide (DMF) has been studied. The sublines (termed lines 66 and 67) differ significantly in their single dose survival responses to x-irradiation. Survival responses were fitted to the single-hit, multi-target, and linear-quadratic equations. For non-DMF treated cells, line 67 radiation parameters were: D 0 ( Gy ) = 1.41, D q ( Gy ) = 1.41, n (extrapolation number) = 2.73, α (10 −1 Gy −1 ) = 2.83, and β (10 −2 Gy −2 ) = 4.17. For line 66, these values were D 0 = 1.63, D g = 3.01, n = 6.4, α = 0.45, and β = 4.28. For investigation of DMF effects, cells were seeded at 1 x 10 4 per 25 cm 2 tissue culture flask in a DMF concentration of 0.8 % (0.10 M) and allowed to multiply for 4 days. For line 67, this increased the cell culture doubling time by about 27 %, and for line 66, by about 22 %. Cells were still in exponential growth at the time of irradiation. Immediately prior to irradiation, the media was removed, fresh media added and flasks were exposed to 100 kVp X rays. After exposure, cells were removed by trypsinization, counted and replated at appropriate dilution in fresh media. The survival parameters for line 67 (the radiosensitive line) then became D 0) = 1.56, D q = 0.89, n = 1.77 , α = 3.39, and β = 3.49. For line 66 (the radioresistant line), the survival parameters were D 0 = 1.56, D q , = 0.94, n = 1.83, α = 4.14, and β = 2.41. These results indicate that DMF may have the potential for modifying the low dose “shoulder” region of survival, particularly for cell lines that possess large shoulders.

Response of a human colon adenocarcinoma (DLD-1) to X irradiation and mitomycin C in vivo

Mice hosting a heterogeneous human colon xenograft tumor produced by subcutaneous injection of the DLD-1 tumor cell line were treated either with x irradiation alone, with mitomycin C alone (4 mg/kg), or with x irradiation given two hours after intraperitoneal injection of mitomycin C (4 mg/kg). Radiation alone produced a dose dependent delay in the time needed for tumors to regrow to twice their size at the time of irradiation, and in the mice receiving mitomycin C plus x irradiation, an additional growth delay equivalent to that produced by 3-3.5 Gy of X rays was seen at all X ray dose levels. As the DLD-1 tumor xenografts do not appear to possess a significant hypoxic fraction, we conclude that the two agents are acting in a simple additive cytotoxic manner by the killing of oxic tumor cells.

Responses of oxic and hypoxic human colon tumor cells to hyperthermia.

In this research, a subpopulation of cells isolated from a colon tumor cell line that was originally established from a human surgical biopsy specimen was examined for its response to hyperthermic ...